Drugs for liver diseases

ABSTRACT

Preventives and/or remedies for liver diseases, which comprise monocyte chemoattractant protein-1 (MCP-1) function inhibitors as active ingredients, respectively. Administration of the MCP-1 function inhibitors brings about effects in preventing and/or remedying liver diseases.

TECHNICAL FIELD

This invention relates to novel drugs for liver diseases, and also to anovel preventive and/or remedial method for liver diseases.

BACKGROUND ART

Chemokines are a group of proteins having migration activity forleukocytes and lymphocytes. From their structures, these chemokines canbe divided roughly into four types. Those with the first and secondcysteines arranged continuously are called “CC chemokines”.

Monocyte chemoattractant protein-1 (MCP-1), one of the CC chemokines,was reported as a protein by itself, and at substantially the same time,its cDNA sequence was ascertained (J. Exp. Med., 169, 1449-1459, 1989;J. Exp. Med., 169, 1485-1490, 1989; FEBS lett., 244, 487-493, 1989).

Receptors which recognize MCP-1 have already been identified, and theircDNAs have also been cloned (Proc. Natl. Acad. Sci. USA, 91, 2752-2756,1994; Biochem. Biophys. Res. Commun., 202, 1156-1162, 1994). Nine typesof receptors are now known as CC chemokine receptors, and the MCP-1receptor is called “CCR2”.

Rollins et al. reported that they prepared a variety of amino acidmutants of MCP-1 protein and some of the amino acid mutants were foundto have lost cell migration activity (J. Biol. Chem., 269, 15918-15924,1994). Among these mutants, the mutant obtained by deleting the secondto eighth amino acids as counted from the N terminal, that is, 7ND-MCP-1has binding ability to CCR2, but does not provoke cell migration. As adominant negative, on the other hand, 7ND-MCP-1 forms a dimer withwild-type MCP-1 and inhibits the function of MCP-1. Further, it is knownthat N-terminal deletions of chemokines are potent dominant negativeinhibitors of chemokine-receptor interaction by forming heterodimerswith the corresponding endogenous monomer of the chemokine and thatthese inhibitors are effective for the remedy of inflammations such aspost-angioplasty restenosis, rheumatoid arthritis, inflammatory boweldisease, multiple sclerosis, and chronic pulmonary inflammation, e.g.,pulmonary fibrosis; autoimmune disease; and the like (JP-A-11506005).

Fibrosis of the liver is a morbidity in which destruction of the normaltissue structure, proliferation of fibroblasts and accumulation ofextracellular matrices advance, and cirrhosis is a post-fibrosisdisease. At present, no effective and safe remedial method has beenestablished yet for these diseases. For example, various symptomatictreatments have been applied to cirrhosis, but cirrhosis advances touncompensated cirrhosis, resulting in poor prognostic improvements.

An object of the present invention is to provide a novel preventiveand/or remedy for a liver disease such as hepatic fibrosis or cirrhosisand further, a novel preventive and/or remedial method for such a liverdisease.

DISCLOSURE OF THE INVENTION

The present inventors have ascertained that 7ND-MCP-1 produced inmyocytes by intramuscular injection of an expression vector containing7ND-MCP-1 gene, into the femoral region of a model animal (rat)suppresses hepatic fibrosis and have found that MCP-1 functioninhibitors are useful as preventives and/or remedies for liver diseases,leading to the completion of the present invention.

Described specifically, the present invention provides a preventiveand/or remedy for a liver disease, comprising an MCP-1 functioninhibitor as an active ingredient.

The present invention also provides a preventive and/or remedial methodfor a liver disease, which comprises administering a gene, which encodesan MCP-1 antagonist or an MCP-1 dominant negative, to an organism.

The present invention further provides a preventive and/or remedycomposition for a liver disease, comprising an MCP-1 function inhibitorand a pharmaceutically acceptable carrier.

The present invention still further provides use of an MCP-1 functioninhibitor for the manufacture of a preventive and/or remedy for a liverdisease.

BEST MODE FOR CARRYING OUT THE INVENTION

No particular limitation is imposed on the MCP-1 function inhibitor foruse in the present invention insofar as it can inhibit the function ofMCP-1 in the organism. Specific examples can includeanti-MCP-1-antibodies (including polyclonals and monoclonals), MCP-1antagonists (including proteins and non-protein, low molecularcompounds), MCP-1 dominant negatives (including proteins andnon-protein, low molecular compounds), and, when those capable ofinhibiting the function of MCP-1 are proteins, also genes encoding suchproteins. As these antibodies, antagonists, dominant negatives, andencoding genes, a variety of antibodies, antagonists, dominant negativesand encoding genes are already known. Further, those available bymethods known per se in the art are all usable in the present invention.

For example, anti-MCP-1 antibodies can be obtained by the proceduredisclosed in J. Immunology, 147, 2229-2233, 1991, while MCP-1antagonists and MCP-1 dominant negatives are known from JP-A-11506005and the like.

In the present invention, introduction of a gene encoding an MCP-1function inhibitor is more preferred than administration of the MCP-1function inhibitor as a protein to an organism, because the formerallows the gene to remain longer in the organism (blood).

In the present invention, MCP-1 antagonists or MCP-1 dominant negativesare preferred, with 7ND-MCP-1 being particularly preferred. Further,genes encoding MCP-1 antagonists or MCP-1 dominant negatives arepreferred, with a gene encoding 7ND-MCP-1 being particularly preferred.As the gene encoding 7ND-MCP-1, DNA having the base sequence indicatedby SEQ ID NO: 1 of the Sequence Listing can be used. This DNA can beprepared by a genetic engineering procedure known per se in the art.Described specifically, from the base sequence of a DNA encoding thewild-type MCP-1 and indicated by SEQ. ID. NO: 2 of the Sequence Listing,the DNA can be prepared using PCR which employs a synthesis primer.

No particular limitation is imposed on an expression vector to be usedfor the expression of the gene in an organism insofar as it can exhibitits function. Illustrative are plasmid vectors such as pcDNA3, pEF-BOSand pXT1; and retrovirus vectors such as adenovirus vectors andSendaivirus vectors. Upon constructing an expression vector, it is alsopossible to use a promoter or an enhancer. No particular limitation isimposed on the promoter or an enhance insofar as it functions in a host(organism). Examples of the promoter can include SV40 promoter, CMVpromoter, HSV-TK, SRα, and RSV.

To have the gene expressed in the host (organism), liposomes are alsousable. In this case, the gene may exist inside the liposomes, or insideor outside the lipid bilayer membranes which constitute the liposomes. Avariety of liposome compositions are known to permit the expression ofthe gene in the host (organism).

To confirm production of 7ND-MCP-1 protein from the 7ND-MCP-1 geneintroduced, it is only necessary to determine by ELISA whether or notthe protein exists in serum.

Administration of the MCP-1 function inhibitor, which is an activeingredient of the preventive and/or remedy according to the presentinvention for a liver disease, to organisms of animals including humanbeing can be conducted orally or parenterally. When the functioninhibitor is a protein, parenteral administration is desired. As aparenteral administration method, injection can be mentioned. Injectioncan be performed directly to a diseased part (the liver) or to a partother than the liver, such as an artery, vein, muscle, skin orsubcutaneous tissue. As a pharmaceutical preparation (preparation forms)for injecting the MCP-1 function inhibitor, an injection can bementioned. This injection can be obtained by known pharmaceuticalpreparation manufacturing technology. Upon manufacturing the injection,one or more of known additives to pharmaceutical preparations can beadded. Illustrative are isotonicities, buffers, preservatives,excipients, and soothing agents.

The dosage to each patient can be adequately determined depending on hisor her condition, age, sex, weight and the like. For example, 0.1 to1,000 mg (in the case of a protein) or 0.01 to 100 mg (in the case of agene) may be administered once in 2 to 4 weeks.

EXAMPLE

The present invention will next be described in further detail based onan example, although the present invention shall by no means be limitedto the example.

(1) Construction and Expression of 7ND-MCP-1

A plasmid vector encoding 7ND-MCP-1 was prepared by PCR using the pCDNA3vector plasmid which encodes MCP-1 as a template. Each mutation wasconfirmed by a DNA sequence analysis from both directions. The resultantPCR product encoding 7ND-MCP-1 was inserted into the multicloning siteof the pcDNA3 vector plasmid, the vector plasmid was transformed inEscherichia coli, and then, the plasmid DNA was purified using “PlasmidGiga Kit” (QIAGEN GmbH).

(2) Effect of 7ND-MCP-1 on Dimethylnitrosamine-Induced Hepatic Fibrosisin Rats

Hepatic fibrotic model rats were prepared by intraperitoneallyadministering 1% dimethylnitrosamine (100 μL/100 g-rat weight) to maleWistar rats daily on three straight days a week for 4 weeks in total.Three days before administration of the mutated MCP-1 gene (7ND-MCP-1gene), 0.25% bupivacaine hydrochloride (100 μL/100 g-rat weight) wasintramuscularly injected to the right femoral muscles of the rats toconduct pretreatment for increased efficiency of gene introduction.

The mutated MCP-1 gene (7ND-MCP-1 gene) was intramuscularly injected(100 μg DNA [1 μg/1 μL]/100 g-rat weight) into the pretreated parts onthe day of start of the dimethylnitrosamine administration. To a controlgroup, the vector DNA was administered in the same amount. The mutatedMCP-1 gene (7ND-MCP-1 gene) was readministered in the same amount asmentioned above to the left femoral muscles of the rats on the 12^(th)day of the dimethylnitrosamine administration. Further, three daysbefore the readministration (on the 12^(th) day of thedimethylnitrosamine administration), the above-mentioned pretreatmentwith bupivacaine hydrochloride was applied likewise. To the controlgroup, the vector DNA was also administered similarly.

Twenty-eight (28) days later, the liver was enucleated, and its weight,and levels of tissue fibrosis and tissue hydroxyproline were measured.The fibrosis level was determined by staining a fibrosed part inaccordance with the Masson's trichrome staining. The level of tissuehydroxyproline was measured by HPLC.

As a result, the liver weight was 10.42±4.01 g (p<0.05 according to theMann-Whitney significant test) and ± in the group administered with themutated MCP-1 gene (7ND-MCP-1 gene) as opposed to 4.95±2.00 g in thecontrol group, and the fibrosis level was ±, as opposed to +++ in thecontrol group. A pronounced hepatic fibrosis inhibiting effect wasobserved.

The tissue hydroxyproline level was 28.2±9.12 μmol/g-rat liver weight(p<0.05 according to the Mann-Whitney significant test) in the groupadministered with the mutated MCP-1 gene (7ND-MCP-1 gene) as opposed to186.75±130.78 μmol/g-rat liver weight) in the control group. Asignificant lowering effect on the hydroxyproline in the liver tissue bythe administration of the mutated MCP-1 gene (7ND-MCP-1 gene) was henceobserved.

Industrial Applicability

As evident from the Example, the MCP-1 function inhibitors are useful aspreventives and/or remedies for liver diseases such as hepatic fibrosisand cirrhosis.

1. A preventive and/or remedy for a liver disease, comprising a monocytechemoattractant protein-1 (MCP-1) function inhibitor as an activeingredient.
 2. A preventive and/or remedy, wherein said MCP-1 functioninhibitor comprises one or more inhibitors selected from anti-MCP-1antibodies, MCP-1 antagonists, MCP-1 dominant negatives and encodinggenes thereof.
 3. A preventive and/or remedy according to claim 2,wherein said genes encoding said MCP-1 dominant negatives are indicatedby SEQ ID NO: 1 of the Sequence Listing.
 4. A preventive and/or remedialmethod for a liver disease, which comprises administering a gene, whichencodes an MCP-1 dominant negative, to an organism.
 5. A preventiveand/or remedial method according to claim 4, wherein said gene isrepresented by SEQ ID NO: 1 of Sequence Listing.
 6. A preventive and/orremedial method according to claim 5, wherein said gene is administeredto said organism at a site other than the liver.
 7. A preventive and/orremedial method according to claim 6, wherein said site other than theliver is a muscle.
 8. A preventive and/or remedy composition for a liverdisease, comprising an MCP-1 function inhibitor and a pharmaceuticallyacceptable carrier.
 9. Use of an MCP-1 function inhibitor for themanufacture of a preventive and/or remedy for a liver disease.